Mapping spatially resolved transcriptomes in human and mouse pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis and limited treatment options. Efforts to identify effective treatments are thwarted by limited understanding of IPF pathogenesis and poor translatability of available preclinical models. Here we generated spatially resolved transcriptome maps of human IPF (n = 4) and bleomycin-induced mouse pulmonary fibrosis (n = 6) to address these limitations. We uncovered distinct fibrotic niches in the IPF lung, characterized by aberrant alveolar epithelial cells in a microenvironment dominated by transforming growth factor beta signaling alongside predicted regulators, such as TP53 and APOE. We also identified a clear divergence between the arrested alveolar regeneration in the IPF fibrotic niches and the active tissue repair in the acutely fibrotic mouse lung. Our study offers in-depth insights into the IPF transcriptional landscape and proposes alveolar regeneration as a promising therapeutic strategy for IPF.

alveolar and fibrotic regions, respectively (Extended Data Fig. 2a,b, Supplementary Table 3).A large overlap of DEGs was observed between these regions, with 223 (95%) upregulated genes and 61 (86%) downregulated genes in IPF alveolar regions also significantly altered in IPF fibrotic regions (Extended Data Fig. 2c).The substantial overlap of DEGs points to a common molecular foundation underlying both "early" and advanced stages of IPF within the same lung.This indicates widespread molecular changes present in the pre-fibrotic alveolar tissue within the IPF lungs, even in the absence of apparent tissue remodeling of the lung parenchyma at a histopathological level.This analysis pinpointed genes of interest in early fibrogenic processes within the alveolar compartment, however, assessing their importance at a spatial level is needed to determine whether they are found adjacent to or distant from to the fibrotic lesions.To delve deeper into the spatial dynamics of the shared upregulated DEGs, we analyzed the correlation between their gene expression and the distance from the fibrotic border into the alveolar regions (Extended Data Fig. 2d (i)).The correlation (Pearson) was computed at the 0-500 µm distance and allowed for selection of significantly correlated genes (FDR adj.p-value < 0.05, Supplementary Table 3).Ten genes had a decreasing expression moving into the alveolar tissue (negatively correlated), and many of them displayed a peaked expression at the fibrosis side of the border (CCL18, CXCL10, CXCL11, IDO1, MT1X, PKP2, RAB31).These genes are largely expressed by cells of myeloid origin and suggests recruitment of immune cells to the edge of fibrotic remodeling.Conversely, 42 DEGs had a positive correlation of gene expression, i.e. increasing expression along the distance from the fibrotic border.
Examining the top 10 positively correlated genes, a decrease in gene expression was seen specifically around the border between fibrotic and alveolar tissue.These genes included AEBP1, APOLD1, COL1A1, COL1A2, LTBP1, and SPARC, and their expression patterns may reflect fibroblast-dense regions and ongoing structural alteration within the transitioning alveolar areas.To confirm the indication of various cell types based on gene expression around the fibrotic border, we inspected the inferred cell densities of myofibroblasts, fibroblasts, monocytes, macrophages, and AT1 cells (Extended Data Fig. 2d bottom panel).This revealed an accumulation of macrophages and monocytes at the fibrotic side of the border, aligning with the peaked expression of the myeloid-related genes.Additionally, the observed decrease of structural gene expression at the interface coincided with lower densities of myofibroblasts and fibroblasts, further suggesting ongoing remodeling and fibroblast activity within the transitional zone.

Supplementary Note 3. Extended description of human NMF factors
Factor 2 (F2).F2 presents a distinct molecular signature, with its activity predominantly observed in the disease state (Fig. 2a).This factor is characterized by a diverse set of genes, including SLC34A2, SFTPB, and CTSH, which are implicated in surfactant metabolism, oxidative stress response, and epithelial cell function.Notably, F2 appears to overlap with areas of honeycombing and fibrosis within IPF lungs, similarly to F5 and F21 (Supplementary Fig. 1a).
The enrichment in pathways such as surfactant metabolism and pulmonary interstitial thickening (Supplementary Table 4) suggests a role in the aberrant alveolar environment of IPF.The overlap of F2 with regions of honeycombing and fibrosis further highlights the association of F2 with advanced disease features, pointing towards a signature that captures genes driving fibrotic remodeling and altering pulmonary surfactant balance.
While both F4 and F14 were highly over-represented in the IPF samples, associated with regions of fibrosis (Fig. 2a), and share several contributing genes (Supplementary Fig. 1b), the distinct differences between them warrant further discussion.F4 was characterized by an array of ECMrelated genes, indicating broad involvement in ECM remodeling and fibrosis, underpinning the structural alterations prevalent in IPF.In contrast, F14 extended beyond the ECM and fibrosis-related gene signature to include keratins such as KRT7 and KRT8, which were not prominently featured in F4.This unique aspect of F14 suggests its potential linkage to the KRT5-/KRT17+ AbBa cell population, a cell type of particular interest due to its potential role in disease progression 2,3 .
The spatial correlation patterns of F4 and F14 with cell densities (Fig. 1e) offer additional insights into their profiles within the IPF tissue.F4 was associated with both fibroblast and myofibroblast densities, highlighting involvement in a range of fibrotic remodeling processes.On the other hand, F14 correlated with myofibroblast densities, without a significant association with fibroblasts, potentially suggesting a more focused involvement linked to the differentiation state of myofibroblasts and their contribution to ECM deposition and tissue stiffening.
Factor 6 (F6).F6 is enriched in genes encoding components of the immunoglobulin complex (IGHA1, IGKC, IGHG series).The presence of genes such as CD79A, a part of the B cell receptor (BCR) complex, along with JCHAIN and MZB1, which are critical for immunoglobulin assembly and secretion, indicates a presence of B cell involvement embedded within the studied IPF lungs.The correlation of F6 with inferred densities of plasma cells in IPF samples (Fig. 1e) moreover points to a contribution of these cells to the immune response within the fibrotic lung environment.This observation is supported by pathway enrichment analyses identifying immunoglobulin complex, complement activation, and B cell receptor signaling pathway among the top significant pathways (Supplementary Table 4), implying an interplay between adaptive immunity and fibrosis in IPF.
F6 activity was often found within regions affected by severe tissue remodeling and may therefore indicate a potential role for the humoral immune response in driving or exacerbating fibrotic remodeling.Whether the presence of B/plasma cell infiltrates contributes to or results from fibrosis, warrants further investigations, as does any considerations for therapeutic strategies aimed at modulating B cell activity to influence IPF progression.
Furthermore, the presence of non-immunoglobulin genes such as hypoxia-induced thioredoxin domain-containing protein 5 (TXNDC5), prostaglandin D2 synthase (PTGDS), and superoxide dismutase 2 (SOD2) within F6 suggests a broader involvement in cellular stress responses, potentially linking immune activation with other pathological processes in the disease.
Factor 9 (F9).F9 was identified as a distinct molecular signature within the more mildly remodeled IPF lung tissues, characterized by an expression pattern not found in healthy controls (Fig. 2a).This factor contains a specific subset of genes associated with oxidative stress, inflammation, ECM remodeling, and vascular changes, suggesting a joint presence of these biological processes, potentially indicative of early-stage fibrotic alterations or protective responses against fibrosis progression.
The spatial co-localization of F9 with endothelial cells and PLIN2+ fibroblasts (Fig. 1e) underscores a metabolic and vascular remodeling state that may represent an adaptive or early pathogenic response within the IPF lung tissue.Taken together, F9 reveals a complex molecular environment potentially representative of an early fibrogenic stage.The composition of genes associated with oxidative stress, inflammation, ECM remodeling, and vascular changes, distinct from more advanced fibrotic niches, highlights the importance of recognizing and understanding these early molecular alterations.

Supplementary Note 4. Expected aberrant basaloid cell detection rate in IPF patients
The detection rate of the KRT5-/KRT17+ AbBa cell type in IPF patients is of value to understand in order to estimate the number of samples needed to detect the cell type.Based on previously published scRNA-seq data from IPF lungs 2,3 , where the AbBa cell type was described, it is apparent that this cell type appears in a large proportion of the studied IPF lungs albeit at low cell count numbers.
The Adams et al. scRNA-seq dataset 2 contains a larger IPF cohort of 32 patients.In this study, they annotated an aberrant basaloid cell type that was detected in almost all IPF patient samples (30/32, i.e. 93.8%; Supplementary Fig. 2b, d).Nonetheless, the percentage of detected aberrant basaloid cells compared to the total sample cell count was under 2% for all patient samples.Our present study is in line with the aforementioned scRNA-seq studies in terms of the KRT5-/KRT17+ AbBa cell type detection rate among IPF donors (marked presence of hsF14 hi -C0 cluster spots in 3/4 donors, i.e. 75% of donors) (Supplementary Fig. 2e).We moreover see a higher abundance of the AbBa hsF14 hi -C0 cluster spots in IPF donor 3, while lower in IPF donors 1 and 4, likely reflecting a diversity in the number of captured AbBa cells within the studied tissue samples.The number of hsF14 hi -C0 cluster spots identified per sample was low, not unexpectedly given the rarity of the AbBa cell type.For reference, the highest number of F14 hi -C0 spots was detected in the IPF donor 3 B1 tissue, where it corresponded to 2.26% of the total number of spots of that tissue section.However, due to differences in input material between studies and the multicellular resolution of a Visium spot, it is important to keep in mind that spot detection rates cannot be used to directly estimate cell type detection rates.